Indicator displacement assays inside live cells

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4. Appendix

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NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1

Pancreatic beta cell death is the fundamental cause of type 1 diabetes (T1D) and a contributing factor to the reduced beta cell mass in type 2 diabetes (T2D)^1–4. In both cases, the mechanisms of beta cell death are complex and as yet not fully defined. Thus, multiple triggering factors have been identified; these factors initiate a variety of signal-ing cascades that affect the expression of apoptotic genes, leadsignal-ing to subsequent beta cell failure. In T1D, autoimmune destruction of insulin-producing beta cells and critically diminished beta cell mass are hallmarks of the disease². Beta cell destruction occurs through immune-mediated processes; mononuclear cell infiltration in the pancreatic islets and interaction between antigen-presenting cells and T cells lead to high local concentrations of inflammatory cytokines, chemokines, reactive oxygen species and other apoptotic triggers (for example, the perforin and Fas–Fas ligand systems)². In T2D, beta cell dysfunction and reduced beta cell mass are the ultimate events leading to the development of clinically overt disease in insulin-resistant indi-viduals. Beta cell destruction is caused by multiple stimuli including glucotoxicity, lipotoxicity, proinflammatory cytokines, endoplasmic reticulum stress and oxidative stress⁵. Unfortunately, although it has been demonstrated that even a small amount of preserved endogenous insulin secretion has great benefits in terms of clinical outcome⁶, none of the currently widely used antidiabetic agents target the maintenance of endogenous beta cell mass.

Beta cells are highly sensitive to apoptotic damages induced by multi-ple stressors such as inflammatory and oxidative assault, owing at least in part to their low expression of cytoprotective enzymes⁷. The initial trigger of beta cell death still remains unclear; it follows an orchestra

of events, which makes the initiation of beta cell death complex and its blockade difficult to successfully achieve in vivo. Therefore, the identification of a common key regulator of beta cell apoptosis would offer a new therapeutic target for the treatment of diabetes.

The identification of the genes that regulate apoptosis has laid the foundation for the discovery of new drug targets. MST1 (also known as STK4 and KRS2) is a ubiquitously expressed serine-threonine kinase that is part of the Hippo signaling pathway and involved in multiple cellular processes such as morphogenesis, proliferation, stress response and apoptosis^8,9. MST1 is a target and activator of caspases, serving to amplify the apoptotic signaling pathway^10,11. Thr183 in subdomain VIII of MST1 has been defined as a primary site for the phosphoactiva-tion and the autophosphorylaphosphoactiva-tion of MST1 and is essential for kinase activation. Both phosphorylation and caspase-mediated cleavage are required for full activation of MST1 during apoptosis. MST1 promotes cell death through regulation of multiple downstream targets such as LATS1 and LATS2, histone H2B and members of the FOXO family, as well as through induction of stress kinase c-Jun-N-terminal kinase (JNK) and activation of caspase-3 (refs. 9,12,13).

Genetic mutations and/or metabolic disturbances can alter protein networks and thereby disrupt downstream signaling pathways that are essential for beta cell survival and function. The transcription factor pancreatic duodenal homeobox-1 (PDX1, previously called IPF1, IDX1, STF1 or IUF1)^14,15 is a key mediator of beta cell development and function¹⁶. In humans, mutations in the PDX1 gene can predispose individuals to develop maturity onset diabetes of the young, type 4 (MODY 4)¹⁷, suggesting a critical role for PDX1 in

1Centre for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany. ²Department of Incretin & Islet Biology, Novo Nordisk A/S, Denmark. ³State Key Laboratory of Genetic Engineering and National Center for International Research of Development and Disease, Institute of Developmental Biology and Molecular Medicine, Fudan University, Shanghai, China. ⁴Division of Transplantation, University of Illinois at Chicago, Chicago, Illinois, USA. ⁵INSERM U859 Biotherapies for Diabetes, European Genomic Institute for Diabetes, Lille Regional Hospital Center, University of Lille, Lille, France. ⁶These authors contributed equally to this work.

Correspondence should be addressed to K.M. (kmaedler@uni-bremen.de) or A.A. (ardestani.amin@gmail.com).

Received 5 December 2013; accepted 21 January 2014; published online 16 March 2014; doi:10.1038/nm.3482

MST1 is a key regulator of beta cell apoptosis and dysfunction in diabetes

Amin Ardestani¹, Federico Paroni^1,6, Zahra Azizi^1,6, Supreet Kaur^1,6, Vrushali Khobragade¹, Ting Yuan¹, Thomas Frogne², Wufan Tao³, Jose Oberholzer⁴, Francois Pattou⁵, Julie Kerr Conte⁵ & Kathrin Maedler¹

Apoptotic cell death is a hallmark of the loss of insulin-producing beta cells in all forms of diabetes mellitus. Current treatments fail to halt the decline in functional beta cell mass, and strategies to prevent beta cell apoptosis and dysfunction are urgently needed. Here, we identified mammalian sterile 20–like kinase-1 (MST1) as a critical regulator of apoptotic beta cell death and function. Under diabetogenic conditions, MST1 was strongly activated in beta cells in human and mouse islets and specifically induced the mitochondrial-dependent pathway of apoptosis through upregulation of the BCL-2 homology-3 (BH3)-only protein BIM. MST1 directly phosphorylated the beta cell transcription factor PDX1 at T11, resulting in the latter’s ubiquitination and degradation and thus in impaired insulin secretion. MST1 deficiency completely restored normoglycemia, beta cell function and survival in vitro and in vivo. We show MST1 as a proapoptotic kinase and key mediator of apoptotic signaling and beta cell dysfunction and suggest that it may serve as target for the development of new therapies for diabetes.

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2 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

mature beta cells; reduced PDX1 expression affects insulin production and secretion and predisposes to beta cell apoptosis^16,18.

Because MST1 acts as common mediator in multiple apoptotic signaling pathways, we hypothesized that it is an initiating trigger of apoptotic signaling in beta cells. MST1 depletion completely restored normogylcemia and insulin secretion and prevented diabetes progres-sion. These findings suggest that MST1 could be a fundamental target for diabetes therapy.

RESULTS

MST1 is activated in diabetes

To test whether MST1 activation is correlated with beta cell apopto-sis, we exposed isolated human and mouse islets and the rat beta cell line INS-1E to a complex diabetogenic milieu. MST1 activity was highly upregulated in these cells under these conditions (created through incubation with the cytokines interleukin-1B (IL-1B) and interferon-G (IFN-G) (IL/IF), upon chronic exposure to increasing glucose concentrations (22.2 and 33.3 mM) or palmitic acid, or upon exposure to acute oxidative stress from hydrogen peroxide) (Fig. 1a–c and Supplementary Fig. 1a,b). The upregulation of MST1 occurred by both caspase-mediated cleavage and autophosphorylation (yield-ing MST1 phosphorylated on T183 (pMST1)). This was accompanied by higher phosphorylation of histone H2B as well as induction of JNK signaling (Fig. 1a–c). In contrast, short-term culture with high glucose concentrations (11.2, 22.2 and 33.3 mM) induced neither apoptosis nor MST1 cleavage and phosphorylation (Supplementary Fig. 1d). MST1 was also activated in islets from subjects with T2D (Fig. 1d), obese diabetic Lepr^db mice (db/db mice, Fig. 1e) and hyperglycemic mice fed with a high-fat, high-sucrose diet (HFD) for 16 weeks (Supplementary Fig. 1c). This activation correlated directly with beta cell apoptosis as described previously¹⁹, as whenever MSTI was induced, apoptosis was also higher. To confirm the beta cell–

specific upregulation of MST1, we performed double immunostaining for pMST1 and insulin in pancreatic islets from subjects with poorly controlled T2D (Fig. 1d) and pancreatic islets from db/db mice (Fig. 1e) and found pMST1 staining in beta cells, whereas there was almost no signal in cells from subjects without diabetes or control mice.

Caspase-3 and JNK act not only as downstream targets but also as upstream activators of MST1 through cleavage- and phosphor-ylation-dependent mechanisms^12,20, and they may initiate a vicious cycle and a proapoptotic signaling cascade in beta cells. Using inhibi-tors of JNK (SP600125) and caspase-3 (z-DEVD-fmk) and siRNA to caspase-3, we found that both JNK and caspase-3 were responsible for stress-induced MST1 cleavage by diabetic stimuli in human islets and INS-1E cells (Supplementary Fig. 1e–h), suggesting that MST1 induces a positive feedback loop with caspase-3 under diabetogenic conditions.

Because phosphatidylinositol-3 kinase (PI3K)-AKT signaling is a key regulator of beta cell survival and function^21,22 and MST1 sig-naling is negatively regulated by this pathway in other cell types^23,24, we hypothesized that AKT is an important negative regulator of MST1. Maintaining AKT activation through either exogenous addi-tion of mitogens such as glucagon-like peptide-1 (GLP1) or insulin or overexpression of constitutively active AKT1 (Myr-AKT1, con-taining a myristoylation sequence and HA tag) inhibited glucose- and cytokine-induced phosphorylation of MST1, MST1 cleavage and apoptosis (Fig. 1f and Supplementary Fig. 2a–d). As GLP1 and insulin exert their cell survival actions primarily through the PI3K-AKT pathway^21,25, we tested whether inhibition of this pro-survival signaling might enhance MST1 activation. PI3K and AKT

were inhibited by LY294002, and triciribine (an AKT inhibitor) led to lower levels of phosphorylation of Gsk3 and Foxo1, two well- characterized AKT substrates, and induced MST1 activation (Fig. 1g,h and Supplementary Fig. 2e). We further corroborated these find-ings using siRNA against AKT, which led to a critical upregulation of MST1 activity and potentiated cytokine-induced phosphoryla-tion of MST1 and beta cell death (Supplementary Fig. 2f). MST1 overexpression also diminished insulin-induced AKT phosphor-ylation and, conversely, there was higher AKT phosphorphosphor-ylation in MST1-depleted beta cells (Fig. 1i). Knockdown of MST1 expression antagonized the apoptotic effect of AKT inactivation in INS-1E cells, implicating endogenous MST1 in the apoptotic mechanism induced by PI3K-AKT inhibition (Supplementary Fig. 2g,h). In summary, these results suggest that MST1 is activated in prodiabetic conditions in vitro and in vivo, antagonized by PI3K-AKT signaling and depend-ent on the JNK- and caspase-induced apoptotic machinery.

MST1 induces beta cell death

MST1 overexpression alone was also sufficient to induce apoptosis in human and rat beta cells (Fig. 2a–c). To investigate pathways that potentially contribute to MST1-induced beta cell apoptosis, we over-expressed MST1 in human islets and rat INS-1E cells through an adenoviral system, which efficiently upregulated MST1, activated JNK and induced beta cell apoptosis, as determined by an increased number of TUNEL-positive beta cells as well as caspase-3 activation and cleavage of poly-(ADP-ribose) polymerase (PARP), a down-stream substrate of caspase-3 (Fig. 2a–c). Previous data proposed a role for the mitochondrial pathway in MST-dependent signal-ing^26,27. Evaluation of established mitochondrial proteins in overexpressing islets and INS-1E cells showed cleavage of the initiator caspase-9, release of cytochrome c, induction of proapoptotic BAX and a decline in antiapoptotic BCL-2 and BCL-xL levels (Fig. 2b,c and Supplementary Fig. 3a), which led to a reduction of BCL-2/BAX and BCL-xL/BAX ratios. Notably, MST1-induced caspase-3 cleavage was reduced by treatment of human islets with the Bax inhibitor peptide V5 (Fig. 2d), which has been shown to promote beta cell survival²⁸; together, these findings emphasize that MST1-induced apoptosis pro-ceeds via the mitochondrial-dependent pathway. We also analyzed the expression of BH3-only proteins as regulators of the intrinsic cell death pathway²⁹. Of these, BIM was robustly induced, whereas other BH3-only protein levels remained unchanged (Fig. 2b,c and Supplementary Fig. 3b). To assess whether kinase activity of MST1 is required for altering mitochondrial-dependent proteins and induction of apoptosis, we overexpressed a kinase-dead mutant of MST1 (K59R;

dominant-negative MST1 (ref. 30)) in human islets. Unlike wild-type (WT) MST1, MST1-K59R did not change the levels of BIM, BAX, BCL-2, BCL-xL and caspase-3 cleavage (Supplementary Fig. 3c).

We next determined whether BIM is a major molecule whose action would override the proapoptotic action of MST1. Indeed, BIM depletion led to a significant reduction of MST1-induced apoptosis in human islets (Fig. 2e,f).

Overexpression of MST1 further potentiated glucose-induced apop-tosis in beta cells in a BIM-dependent manner (Supplementary Fig. 3d).

BIM is regulated by the JNK³¹ and AKT³² signaling pathways. MST1-induced increase in BIM and subsequent caspase-3 cleavage was prevented by JNK inhibition through overexpression of dn-JNK1 (Fig. 2g) or by the JNK inhibitor (Supplementary Fig. 3e), which suggests that MST1 uses JNK signaling to mediate BIM upregula-tion and inducupregula-tion of apoptosis. We confirmed the involvement of AKT in the regulation of MST1-induced apoptosis by overexpressing

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NATURE MEDICINE ADVANCE ONLINE PUBLICATION 3

Cont T2D T2D

pMST1

Insulin

Merge 0

clMST1 pMST1 1 2 3 4

5 *

*

Protein/actin

Cont T2D

a

0

clMST1pMST1pJNK pH2B clC3 1

2 3

5.5 mM 22.2 mM 33.3 mM

**

**

*

*

* *

**

Protein/actin

Human islets Glc (mM):

clMST1

Actin clC3 MST1

pH2B pJNK pMST1

33.3 22.2 5.5

b

clMst1 pMst1pJnk pH2B clC3

**

**

**

**

**

0 1 2 3 4 5

Protein/actin

11.1 mM 11.1-IL/IF 33.3-Pal

Actin Glc (mM):

clMst1

clC3 Mst1

pH2B pJnk pMst1

Mouse islets

11.1 11.1-IL/IF33.3-Pal

clMST1pMST1pJNK pH2B clC3

* *

*

*

*

0 1 2 3 4 5

Protein/tubulin

5.5 mM 5.5-IL/IF 5.5-IL/IF

Tubulin clC3 clMST1 MST1

pH2B pJNK pMST1

5.5

clMst1 pMst1 pGsk3 clC3 0

1 2 3

4 11.1 mM GFP

22.2 mM GFP 22.2 mM Myr-AKT1

*

*

*

*

#

Protein/actin #

# #

f^Myr-AKT1_{Glc (mM)} ^{–+ –+ +–}

pAkt

clC3 Actin clMst1 Mst1

pMst1

pGsk3

11.1 22.2 22.2 GFP

c

clMst1 pJnk pH2B clC3 0

1 2 3 4 5 6

*

*

* * ** *

*

Protein/tubulin

22.2 mM 33.3 mM 11.1 mM

Tubulin INS-1E

Mst1

clC3 pH2B pJnk clMst1

Glc (mM): 11.1 22.2 33.3

clMst1pJnkpH2B clC3

*

*

*

*

0 1 2 3 4 5 6

Protein/actin

11.1 mM 11.1-IL/IF

Actin clC3 Mst1

pH2B pJnk clMst1

g

clMst1 pAkt

pGsk3pFoxo1 clC3 0

1 2

3 *

*

* *

*

Protein/actin

Cont LY Mst1

LY – +

clMst1 pAkt

pGsk3 pFoxo1 clC3

Actin

d

Actin pMST1 clMST1 MST1

Cont T2D Cont T2D

e

0 1 2 3

clMst1 pMst1

*

*

Protein/actin

db/+ db/db db/+ db/db

pMst1

Insulin

db/db

Merge Actin

pMst1 clMst1 Mst1

db/db db/db db/db

db/+ db/+

h

clMst1 pAkt

pGsk3pFoxo1 clC3

*

*

* *

*

0 1 9

Protein/actin

Cont AKTi

pFoxo1

Actin pAkt pGsk3 clMst1

clC3

AKTi – + i

Insulin

GFP MST1 shScr shMst1

– + – + – + – +

Mst1 pAkt tAkt Actin 11.1 11.1-IL/IF

Figure 1 MST1 is activated in diabetes.

(a–c) Activated MST1 (cleaved (clMST1) and phosphorylated) in human (a) and mouse (b) islets and INS-1E cells (c) exposed to diabetogenic conditions (IL/IF, 22.2 mM glucose, 33.3 mM glucose, or a mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3-Pal)

for 72 h). Western blots of MST1, pMST1, pJNK, pH2B and caspase-3 cleavage (clC3) and densitometry analyses are shown. Cont, control; Glc, glucose. (d,e) Activated MST1 in islets. Human isolated islets from nondiabetic control subjects (n = 7) and subjects with T2D (n = 4, all with documented fasting plasma glucose >150 mg/dl) (d) and from 10-week-old diabetic db/db mice (n = 5) and their heterozygous littermates (db/+, n = 5) (e). Left, western blots of MST1 and pMST1 and densitometry analyses. Right, double immunostaining for pMST1 (red) and insulin (green) in sections from human isolated islets from nondiabetic control subjects and subjects with T2D and from 6-week-old diabetic db/db mice (representative analyses from 10 pancreases from subjects with T2D and >10 pancreases from control subjects and from 7 db/db mice and 7 heterozygous controls are shown). Scale bar, 100 Mm. (f) Western blots of Mst1, clMst1, pMst1, pAkt, pGsk3 and caspase-3 cleavage and densitometry analysis for INS-1E cells transfected with GFP control or Myr-AKT1 expression plasmids exposed to 22.2 mM glucose for 72 h. (g) Western blots of Mst1, clMst1, pAkt, pGsk3, pFoxo1 and caspase-3 cleavage and densitometry analysis for INS-1E cells exposed to the PI3K inhibitor LY294002 (LY, 10 MM for 8 h). (h) Western blots of clMst1, pAkt, pGsk3, pFoxo1 and caspase-3 cleavage and densitometry analysis for INS-1E cells exposed to the AKT inhibitor triciribine (AKTi, 10 MM for 6 h). (i) Western blots of Mst1, pAkt and tAkt for INS-1E cells infected with an adenovirus expressing GFP (Ad-GFP) or MST1 (Ad-MST1) or transfected with shMst1 or shScr control expression plasmids for 48 h, serum-starved for 12 h and then stimulated with insulin. All graphs show densitometry analyses from at least 3 independent experiments normalized to actin or tubulin. All western blots show representative results from at least 3 independent experiments from 3 different donors or mice. Tubulin or actin was used as loading control. Data are expressed as means o s.e.m.

*P < 0.05 compared to untreated or nondiabetic control. ^#P < 0.05 Myr-AKT1 compared to GFP at 22.2 mM glucose.

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4 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

both MST1 and Myr-AKT1, which reduced BIM induction and caspase-3 cleavage (Fig. 2h), indicating that AKT negatively regu-lates the downstream target of MST1. These data suggest that MST1 is a critical mediator of beta cell apoptosis through activation of the BIM-dependent intrinsic apoptotic pathway and controlled by AKT and JNK signaling pathways.

MST1 impairs beta cell function by destabilizing PDX1

We hypothesized that MST1 activation may elicit changes in beta cell–

specific gene transcription that initiate the process of beta cell failure.

Overexpression of MST1 led to a complete loss of glucose-stimulated insulin secretion (GSIS; Fig. 3a and Supplementary Fig. 4a), which could not be accounted for solely by the induction of apoptosis.

Previously, we noted that the critical beta cell transcription factor PDX1, which mediates glucose-induced insulin gene transcription in mature beta cells^16,18, is mislocalized and reduced in diabetes¹⁹. These changes are subsequently associated with impaired beta cell function and hyperglycemia. Stress-induced kinases such as JNK and glycogen synthase kinase-3 (GSK3) phosphorylate PDX1, antagonizing its activity^33,34, which leads to beta cell failure. Thus, we hypothesized

that the drastic reduction in insulin secretion following MST1 over-expression may be mediated by PDX1. PDX1 levels were markedly reduced in response to MST1 overexpression in human islets (Fig. 3b) and INS-1E cells (Supplementary Fig. 4b). In contrast, MST1 overex-pression did not affect the amount of mRNA encoding PDX1 (Fig. 3b and Supplementary Fig. 4b), suggesting that MST1 may regulate PDX1 at the post-transcriptional level. Analysis of PDX1 target genes demonstrated that overexpression of MST1 significantly down-regulated INS (Ins1 or Ins2 for INS-1E), SLC2A2 and GCK in human islets (Fig. 3b) and INS-1E cells (Supplementary Fig. 4b). Although SLC2A2 is not the predominant glucose transporter in human beta cells³⁵, we analyzed its expression to provide comparison to the mouse data.

To gain better insight into the role of MST1 in regulation of insulin secretion, we performed GSIS using two insulin secretagogues: GLP1 and glibenclamide. MST1 overexpression significantly abolished GLP1-enhanced glucose-induced insulin secretion (P < 0.05 compared to control condition), whereas glibenclamide-induced insulin secre-tion was not affected, suggesting that defective insulin secresecre-tion may occur at a step upstream of calcium influx (Supplementary Fig. 4c).

a _TUNEL

Insulin DAPI

MST1 Cont

0 1

Cont GFP MST1

% TUNEL+ beta cells

*

b Human islets c

clMST1

BIM

BAX BCL-2

clC3 Actin clC9

GFP MST1

BCL-xL pMST1

pJNK MST1

0 1 2 3 4

Ad-GFP Ad-MST1

Protein/actin

*

*

*

* *

*

*

BlMpJNK BAXBCL-2BCL-xL clC9 clC3

INS-1E

Bim

Bax Bcl-2

clPARP Actin clC9

GFP MST1 MST1

clC3

0 1 2

3 Ad-GFP

Ad-MST1

Protein/actin

*

*

*

* *

*

Bim Bax Bcl-2 clC9 clC3 clPARP

NC

GFP MST1 GFP MST1 V5

clC3 Actin

0 2 4 6 8

Ad-GFP Ad-MST1

clC3/actin

NC

*

§

V5

d e

0 1 2

siScr siBIM

% TUNEL+ beta cells GFP

*

**

MST1

f

MST1GFPMST1 GFP

siScr siBIM

clC3 clPARP Actin BIM MST1

0 1 2 3

Ad-GFP Ad-MST1 Ad-GFP–siBIM Ad-MST1–siBIM

Protein/actin

*

**

*

**

*

**

BIM clC3 clPARP

g

GFP MST1

BIM clC3 Actin pc-Jun

dn-JNK1 – + – +

0 1 2 3

Ad-GFP Ad-GFP–dn-JNK1 Ad-MST1 Ad-MST1–dn-JNK1

Protein/actin

*

#

*

*

# #

pc-Jun BIM clC3

h

GFP MST1 BIM

clC3 Actin

Myr-AKT1 – + – +

0 1 2 3

Ad-GFP Ad-GFP–AKT1 Ad-MST1 Ad-MST1–AKT1

BIM

Protein/actin

*

+

*

+

clC3 Figure 2 MST1 induces beta cell death. (a–d) MST1 overexpression in human islets (a,b) and INS-1E cells (c) for 48 h. Triple staining for

DAPI (blue), TUNEL (red) and insulin (green) (a) TUNEL analysis from an average number of 18,501 insulin-positive beta cells. Scale bar, 100 Mm.

(b,c) Adenovirus-mediated upregulation of MST1. Western blots and densitometry analysis of MST1, cleaved MST1, BIM, pJNK, BAX, BCL-2, BCL-xL, cleaved caspase-9 (clC9), cleaved caspase-3 and PARP in human islets (b) and INS-1E cells (c). (d) Western blot and densitometry analysis of caspase-3 cleavage from Ad-GFP– or Ad-MST1–infected human islets exposed to BAX inhibitory peptide V5 or negative control (NC) peptide for 36 h. (e,f) Human islets transfected with BIM siRNA (siBIM) or control Scr siRNA (siScr) infected with Ad-GFP or Ad-MST1 for 48 h. (e) Analysis of cells positive for both TUNEL and insulin out of 10,378 insulin-positive counted beta cells. (f) Western blot and densitometry analysis of MST1, BIM and caspase-3 and PARP cleavage. (g) Western blot and densitometry analysis of pc-Jun, BIM and caspase-3 cleavage from human islets transfected with GFP or dn-JNK1 expression plasmids and infected with Ad-GFP or Ad-MST1 for 48 h. (h) Western blot and densitometry analysis of BIM and caspase-3 cleavage from human islets transfected with GFP or Myr-AKT1 expression plasmids and infected with Ad-GFP or Ad-MST1 for 48 h. All graphs show densitometry analysis from at least 3 independent experiments normalized to actin. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Actin was used as loading control. TUNEL analyses are from 3 independent experiments from 3 different donors. Data are expressed as means o s.e.m. *P < 0.05 MST1 overexpression compared to GFP control, §P < 0.05 V5-MST1 compared to MST1, **P < 0.05 siBIM-MST1 compared to siScr-MST1, ^#P < 0.05 dn-JNK–MST1 compared to MST1, ⁺P < 0.05 AKT1-MST1 compared to MST1.

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NATURE MEDICINE ADVANCE ONLINE PUBLICATION 5

MST1 overexpression had no effect on insulin content (Fig. 3a and Supplementary Fig. 4a), and thus insulin secretion was normalized on insulin content.

To clarify the mechanism by which MST1 regulates PDX1, we examined the effects of ectopic expression of MST1 and PDX1 in human embryonic kidney (HEK) 293 cells. We found lower PDX1

levels in cells co-overexpressing WT MST1, whereas the kinase-dead MST1-K59R had no effect (Fig. 3c). Thus, kinase activity is required for MST1-induced PDX1 degradation. Overexpression of MST1 also attenuated the transcriptional activity of PDX1 on the rat insulin pro-moter (RIP), as shown by luciferase assays in PDX1-overexpressing HEK 293 and INS-1E cells (Supplementary Fig. 4d).

+ + –

+ + +

+ + + – – – PDX1

Actin GFP-PDX1 Myc-MST1 CHX

+ + + +

+ + + + – –

– – PDX1 Tubulin GFP-PDX1 Myc-MST1 MG1-32

g IP: PDX1

+ –

+ +

+ + + –

– – –

– PDX1-WT

PDX1-T11A rGST-MST1 pMST1 pT11PDX1 pThr

CHX (h)

PDX1-WT PDX1-T11A MST1 OE

0 PDX1 Tubulin

3 6 9 0 3 6 9 0

2 4

GFPMST1

*

Stimulatory index

0 200 400 600 800 1,000

Insulin content (μU/μg protein)

GFPMST1

+ + +

– –

– – + +

PDX1/tubulin

1

GFP-PDX1 Myc-MST1 dn-MST1 (K59R)

0

0 1 PDX1

Actin

GFP MST1

b

GFPMST1

PDX1/actin

*

c

GFP-PDX1

HEK 293 cells

+ +

+

– –

– –

+ + PDX1

MST1 Tubulin Myc-MST1 dn-MST1 (K59R)

h

+ +

+ –

– –

– – – GFP

PDX1 Tubulin PDX1-WT PDX1-T11A +

– + +

+ + + –

– – –

– PDX1-WT

PDX1-T11A Myc-MST1

MST1 PDX1 Actin

f

+ +

+ + –

– rMST1

pMST1

MST1 pPDX1

PDX1 rPDX1

e

GFP-PDX1

InputIP: MycIP: GFP

+ +

+ – Myc-MST1 IB: Myc-clMST1

IB: GFP IB: GFP

IB: GFP IB: Myc IB: Myc 0

200 400

Basal Stimulated

Insulin to content (% basal control)

GFP MST1

* *

a

0 1

GFP MST1-OE

mRNA to tubulin (normalized to GFP)

* * *

PDX1SLC2A2 GCK INS

PDX1-WT

*

**

PDX1-T11A 6

Stimulatory index

4 2 0

MST1

GFP j

PDX1-WT PDX1-WT + MST1

* *

*

** **

**

PDX1-T11A PDX1-T11A + MST1

SLC2A2 GCK INS

1

mRNA/tubulin/normalized to PDX-WT 0 GFP

IB: PDX1 Human islets

IP: PDX1

IB: Ub PDX1-Ub

+ +

+ + +

+ + + + +

+ +

–

– –

– MST1 HA-ubiquitin MG-132 +

+ + –

d

GFP-PDX1 HEK 293 cells

IP: PDX1

IB: Ub IgGPDX1-Ub + +

+ + + – – – Myc-MST1 MST1 (K59R) HA-ubiquitin

MG-132 + + +

Time (h) 100

PDX1WT

PDX1 remaining (%) PDX1T11A

0 3 6 9

80 60 40 20 0

i _Basal

GFP MST1 GFP MST1 PDX1-WT

*

**

PDX1-T11A 1,000

Insulin to content (% basal control) 800 600 400 200 0

Stimulated

Figure 3 MST1 impairs beta cell function by destabilizing PDX1. (a,b) Adenovirus-mediated GFP or MST1 overexpression in human islets for 96 h. (a) Insulin secretion during 1-h incubation with 2.8 mM (basal) and 16.7 mM (stimulated) glucose, normalized to insulin content and basal secretion at GFP control. The insulin stimulatory index denotes the ratio of secreted insulin during 1-h incubation with 16.7 mM to that secreted during 1-h incubation with 2.8 mM glucose. Insulin content analyzed after GSIS and normalized to whole islet protein is also shown.

(b) Left, western blot and densitometry analysis of PDX1. Right, RT-PCR analysis of PDX1 target genes SLC2A2, GCK and INS. (c) Western blot and densitometry analysis of PDX1 and MST1 from HEK 293 cells transfected with plasmids encoding Myc-MST1 and GFP-PDX1 with kinase-dead MST1 (dn-MST1, K59R) cotransfected with GFP-PDX1 (left) and western blot of PDX-1 from HEK 293 cells treated with CHX for 8 h at 48 h after transfection, (middle) or treated with the proteasome inhibitor MG-132 for 6 h at 36 h after transfection (right). (d) Immunoblotting with ubiquitin-specific antibody after immunoprecipitation with an anti-PDX1 antibody of HEK 293 cells transfected with GFP-PDX1 and hemagglutinin (HA)-ubiquitin (Ub), alone or together with Myc-MST1 or MST1-K59R expression plasmids for 48 h (left) and human islets transfected with HA-ubiquitin and infected with Ad-GFP or Ad-MST1 for 48 h (right; 2 different donors). MG-132 was added during the last 6 h of the experiment. (e) Western blot analysis for Myc and GFP with precipitates and input fraction after reciprocal co-immunoprecipitations (using anti-GFP and anti-Myc antibodies) from HEK 293 cells transfected with GFP-PDX1 alone or together with Myc-MST1 for 48 h. (f) Western blot of pThr (pan–phosphorylated threonine), MST1 and PDX1 from in vitro kinase assay performed with recombinant MST1 (rMST1) and recombinant PDX1 (rPDX1) proteins. (g) Left, western blot of pPDX1 (pT11PDX1) and pThr (after immunoprecipitation with anti-PDX1) from HEK 293 cells transfected with PDX1-WT or PDX1-T11A expression plasmids and subjected to an in vitro kinase assay using recombinant MST1. Middle, western blot of MST1 and PDX1 from HEK 293 cells transfected with WT or PDX1-T11A expression plasmids alone or together with MST1 expression plasmids for 48 h. Right, western blot of PDX1 and densitometry analysis of bands for PDX1-WT or PDX1-T11A cotransfected with MST1 in HEK 293 cells for 36 h and treated with CHX. (h) Western blot of PDX1 from human islets transfected with GFP, PDX1-WT or PDX1-T11A expression plasmids. (i) Insulin secretion during 1-h incubation with 2.8 mM (basal) and 16.7 mM (stimulated) glucose, normalized to insulin content and basal secretion at control and insulin stimulatory index, which denotes the ratio of secreted insulin during 1-h incubation with 16.7 mM to that secreted at 2.8 mM glucose. (j) PDX1 target genes in human islets analyzed by RT-PCR and levels normalized to tubulin and shown as change from PDX1-WT transfected islets from human islets infected with Ad-GFP or Ad-MST1 for 72 h. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Tubulin or actin was used as loading control. RT-PCR (b,j) and GSIS (a,i) show pooled results from 3 independent experiments from 3 different donors. Data are expressed as means o s.e.m.

*P < 0.05 MST overexpression compared to control, **P < 0.05 PDX1-T11A + MST1 compared to PDX1-WT + MST1.

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6 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

To discriminate between a transcriptional or translational and a post-translational effect of MST1 on PDX1, we followed the stability of overexpressed PDX1 upon treatment with cycloheximide (CHX), an inhibitor of protein translation. Upon CHX exposure, PDX1 protein levels rapidly decreased when coexpressed with MST1 (Fig. 3c), which suggests that MST1 reduced PDX1 protein stability. Consistent with these observations, MST1 overexpression also decreased protein stability of endogenous PDX1 in human islets (Supplementary Fig. 4e). In contrast, treatment of PDX1-overexpressing HEK 293 cells with the proteasome inhibitor MG-132 reduced the disappearance of PDX1 (Fig. 3c), indicating that MST1 induced activation of the ubiquitin proteasome pathway.

Proteasomal degradation of PDX1 has been described before and leads to impaired beta cell function and survival³⁶.

We next performed in vivo ubiquitination assays to determine whether MST1 induces PDX1 ubiquitination. PDX1 cotransfected with MST1, but not with MST1-K59R, was heavily ubiquitinated in HEK 293 cells. We confirmed this in human islets by showing that MST1 overexpression strongly promoted endogenous PDX1 ubiquitination (Fig. 3d). Subsequently, we verified a direct interaction between PDX1 and MST1 proteins. Reciprocal co-immunoprecipitations showed the interaction between MST1 and PDX1 in HEK 293 cells cotransfected with GFP-tagged PDX1 and Myc-tagged MST1 (Fig. 3e).

We next examined whether a prodiabetic milieu regulates the asso-ciation between MST1 and PDX1. Notably, both cytokine toxicity and glucotoxicity augment the interaction between MST1 and PDX1 in INS-1E cells (Supplementary Fig. 4e). As we observed that PDX1 ubiquitination and degradation required MST1 kinase activity, we tested whether MST1 directly phosphorylates PDX1. In vitro kinase assays showed that MST1 efficiently phosphorylated PDX1; these included autoradiography using radiolabeled ³²P (Supplementary Fig. 4f), as well as nonradioactive kinase assays and western blotting using an antibody specific to pan–phosphorylated threonine (Fig. 3f). We con-firmed the in vitro kinase assays in HEK 293 cells; coexpression of MST1 and PDX1 led to PDX1 phosphorylation (Supplementary Fig. 4f).

Together, these results establish PDX1 as a substrate for MST1.

We determined the potential MST1-targeted phosphorylation sites on PDX1 theoretically with the NetPhos 2.0 program³⁷. This identified six candidate sites within the PDX1 sequence; T11, T126, T152, T155, T214 and T231 (based on the probability that a phosphosite is a substrate of MST1, given as relative score) (Supplementary Fig. 4g). These six sites were individually mutated to alanine to generate phosphodeficient constructs as described previously³⁸. We subcloned them into pGEX bacterial expression vectors. PDX1-GST fusion proteins with the six different PDX1 mutations were purified from bacteria and used as sub-strates for MST1 in the kinase assay. With the exception of PDX1-T11A, WT recombinant PDX1 and the other mutants proteins were efficiently phosphorylated at threonine (Supplementary Fig. 4h). To confirm this, we transfected all PDX1 mutant plasmids into HEK 293 cells, immuno-precipitated them with a PDX1-specific antibody and incubated them with recombinant MST1 in a kinase assay. MST1 highly phosphorylated PDX1-WT and other mutant proteins, but phosphorylation in the PDX1-T11A mutant was markedly lower (data not shown), indicating that T11 is the major site of phosphorylation by MST1.

In order to confirm T11 as the specific phosphorylation site, we used a phosphospecific antibody against the T11 phosphorylation site in PDX1, which recognized T11 phosphorylation after co-incubation of recombinant PDX1-GST fusion protein with recombinant GST-MST1 (Supplementary Fig. 4h). Consistent with this, co-incubation of immunoprecipitated PDX1-WT or PDX1-T11A with recombinant

MST1 resulted in robust MST1-induced PDX1-WT phosphorylation at the T11 site (shown by antibody to pT11) and in overall threonine phosphorylation (shown by antibody to pan–phosphorylated threo-nine); T11 phosphorylation was markedly reduced in the PDX1-T11A mutant protein (Fig. 3g). We further corroborated this by an in vivo kinase assay (Supplementary Fig. 4h). Alignment of the amino acid sequences of PDX1 from different species revealed that the T11 site is highly conserved among those species (Supplementary Fig. 4i).

If T11 is the specific MST1-induced phosphorylation site of PDX1 and is responsible for beta cell dysfunction, one would expect that mutated PDX1-T11A would reverse beta cell dysfunction. MST1 induced a rapid degradation of exogenous WT PDX1 in the presence of CHX that did not occur in PDX1-T11A mutant–transfected cells (Fig. 3g). Furthermore, the half-life of the PDX1-T11A mutant was similar to that of PDX1-WT in the absence of MST1 (data not shown).

Consistent with these data, there was less PDX1 ubiquitination in the PDX1-T11A–transfected cells than in those transfected with PDX1-WT (Supplementary Fig. 4j).

Because T11 is located within the transactivational domain of PDX1 and to evaluate the functional significance of the T11-dependent ubiquitination and degradation, we assessed transcriptional activity of PDX1. Reduction of PDX1 transcriptional activity occurred only in PDX1-WT– but not in PDX1-T11A–transfected cells (Supplementary Fig. 4j). As the T11A mutation of PDX1 prolongs PDX1 stability in the presence of MST1, we asked whether PDX1 stability is directly linked to improved beta cell function. PDX1-T11A mutant overexpression (Fig. 3h) normalized MST1-induced impairment in GSIS in human islets (Fig. 3i) and INS-1E cells (Supplementary Fig. 4j) and restored MST1-induced downregulation of PDX1 target genes (Fig. 3j and Supplementary Fig. 4j). These findings indicate that MST1-induced PDX1 phosphorylation at T11 leads directly to PDX1 destabilization and impaired beta cell function and suggest that PDX1 is a crucial target of MST1 in the regulation of beta cell function.

MST1 deficiency improves beta cell survival and function Further analyses aimed to prove whether MST1 not only medi-ated beta cell death and impaired function in vitro but also, when downregulated, allowed for rescue from beta cell failure (Fig. 4 and Supplementary Fig. 5). First, about 80% depletion of MST1 in human islets, achieved with siRNA, protected from cytokine and hydrogen peroxide toxicity as well as glucolipotoxicity; beta cell apoptosis was also inhibited (Fig. 4a,b and Supplementary Fig. 5a).

Silencing of MST1 also significantly reduced BIM upregulation induced by diabetogenic conditions in human islets (Fig. 4b,c and Supplementary Fig. 5a).

Second, beta cell function was greatly improved by MST1 gene silencing under diabetogenic conditions (Fig. 4d and Supplementary Fig. 5). Notably, IL/IF- and high glucose + palmitate (HG/Pal)-induced cleavage of caspase-3 and caspase-9 and phosphorylation of H2B was lower in MST1-depleted human islets than in control islets (Fig. 4b). Mst1^−/− islets were largely resistant to IL/IF- and HG/Pal-mediated apoptosis, as determined by TUNEL staining (Fig. 4e). In addition to the protective effect of Mst1 knockout on beta cell survival, Mst1^−/− islets also showed improved GSIS after long-term culture with IL/IF and HG/Pal (Fig. 4f and Supplementary Fig. 5). To further support the role of MST1 as a main mediator of apoptosis in beta cells, we generated INS-1E cells stably transfected with vectors carrying Mst1-targeting shRNA (shMst1) or scram-bled control shRNA (shScr) and found that Mst1 expression in cells stably expressing shMst1 was about 70% lower than that in cells

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NATURE MEDICINE ADVANCE ONLINE PUBLICATION 7

expressing shScr (Fig. 4g). We treated INS-1E clones with IL/IF and HG for 72 h. Bim induction, caspase-3 and PARP cleavage in Mst1-depleted cells were significantly lower than that in control cells (Fig. 4g). Additionally, Mst1 silencing also abrogated caspase-3 and PARP cleavage induced by palmitate (Supplementary Fig. 5b) and hydrogen peroxide (Supplementary Fig. 5c). Cytochrome c release was markedly reduced in Mst1-depleted beta cells under diabetogenic conditions (Supplementary Fig. 5d,e). A second shRNA clone tar-geting the Mst1 gene with comparable gene silencing efficiency con-firmed the antiapoptotic effect of Mst1 silencing in INS-1E cells;

Mst1 depletion markedly suppressed IL/IF- and HG-induced Bim upregulation and cleavage of caspase-3 and PARP (Supplementary

Fig. 5f). Confirmation the results of the shMst1 approach, inhibi-tion of endogenous Mst1 activity by overexpression of Mst1-K59R completely inhibited glucose-induced caspase-3 and PARP cleavage in beta cells (Supplementary Fig. 5g).

Mst1 deficiency significantly attenuated Pdx1 depletion caused by cytokine or high glucose treatment (Fig. 4g and Supplementary Fig. 5f), implying that MST1 is indispensable for the reduction in amount of PDX1 induced by a diabetic milieu. Our next objective was to determine whether knockdown of Mst1 expression leads to improvement of GSIS and restoration of Pdx1 target genes in INS-1E cells under diabetogenic conditions. GSIS was significantly improved in Mst1-depleted beta cells (Fig. 4h and Supplementary Fig. 5j),

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Bcl2L11 to tubulin mRNA (normalized to control)

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shScr shScr 33.3 mM shMst1

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shMst1 33.3 mM

Gck Ins1 Ins2

Figure 4 MST1 deficiency improves beta cell survival and function. (a–d) Analysis of human islets transfected with MST1 siRNA (siMST1) or control siScr and treated with the cytokine mixture IL/IF, 33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate for 72 h.

(a) TUNEL-positive beta cells from an average number of 11,390 insulin-positive beta cells for each treatment condition.

(b) Western blots and densitometry analysis of MST1, pMST1, BIM, pH2B and caspase-9 and caspase-3 cleavage. (c) RT-PCR for BCL2L11 in human islets normalized to tubulin shown as

change from siScr control transfected islets. (d) Insulin stimulatory index denotes the ratio of secreted insulin during 1-h incubation with 16.7 mM to that secreted during 1-h incubation with 2.8 mM glucose (after the indicated treatments). (e) Analysis of TUNEL-positive beta cells from an average number of 24,180 insulin-positive beta cells counted for each treatment condition. (f) Insulin stimulatory index denotes the ratio of secreted insulin during 1-h incubation with 16.7 mM to that secreted during 1-h incubation with 2.8 mM glucose from isolated islets from Mst1^−/− mice and their WT littermates after exposure to the cytokine mixture IL/IF or the mixture of 33.3 mM glucose and 0.5 mM palmitate for 72 h. (g–i) Western blots and densitometry analysis of Mst1, clMst1, pMST1, Bim, Pdx1, caspase-3 and PARP cleavage (g), insulin stimulatory index (h) and RT-PCR analysis of PDX1 target genes Slc2a2, Gck, Ins1 and Ins2 normalized to tubulin and shown as change from shScr (i) in stable INS-1E clones generated by transfection of vectors for shMst1 and shScr control and treated with the cytokine mixture IL/IF or with 22.2 or 33.3 mM glucose for 72 h.

Representative results from 3 independent experiments from 3 different donors (human islets) (b,g). Actin was used as loading control. TUNEL data (a,e), GSIS (d,f,h) or RT-PCR (c,i) show pooled results from 3 independent experiments from 3 different donors (human islets). Data are expressed as means o s.e.m. *P < 0.05 compared to siScr (a–d), WT (e,f) or shScr untreated controls (g,h,i); **P < 0.05 compared to siScr (a–d), WT (e,f) or shScr (g–i) at the same treatment conditions.

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8 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

whereas levels of Pdx1 target genes, for example, Slc2a2, Gck, Ins1 and Ins2, were restored in Mst1-depleted INS-1E cells (Fig. 4i). These data prove MST1 as determinant for beta cell apoptosis and defective insulin secretion under a diabetic milieu in beta cells in vitro.

Mst1 deletion protects from streptozotocin-induced diabetes As MST1 depletion protected from beta cell apoptosis and restored beta cell function in vitro, we hypothesized that Mst1 deficiency may protect against diabetes in vivo by promoting beta cell survival and

preserving beta cell mass. To test this hypothesis, we used Mst1^−/−

mice. Neither body weight nor food intake differed between Mst1^−/−

mice and their WT (Mst1^+/+) littermates (data not shown). Also, glucose tolerance, insulin tolerance and glucose-induced insulin response did not differ between WT and Mst1^−/− mice at 2 months of age (Supplementary Fig. 6a). However, intraperitoneal (i.p.) glucose tolerance tests (GTTs) and i.p. insulin tolerance tests (ITTs) revealed slight improvement in Mst1^−/− mice at 6 months of age at 60 min after glucose or insulin injection (Supplementary Fig. 6b).

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Figure 5 Mst1 deletion protects from diabetes in vivo. (a–g) Mst1^−/−

mice (n = 15) and their WT littermates (n = 14) injected with 40 mg per kg body weight STZ for five consecutive days.

(a) Random fed blood glucose measurements after last STZ injection (day 0) over 21 d and

i.p. GTT (ipGTT) performed at day 17. (b) Insulin secretion during an i.p. GTT measured before (0 min) and 30 min after glucose injection (left) expressed as ratio of secreted insulin at 30 min to that secreted at 0 min (stimulatory index, right). (c) Ratio of secreted insulin and glucose calculated at fed state. (d) Beta cell mass and quantitative analyses from triple stainings for TUNEL or Ki-67, insulin and DAPI expressed as percentage of TUNEL- or Ki-67–positive beta cells o s.e.m. from a mean scored number of 23,121 beta cells for each treatment condition. (e) Percentage of alpha cells (stained in red) and beta cells (stained in green) of the whole pancreatic sections from 10 sections spanning the width of the pancreas. Scale bar, 100 Mm. (f,g) Representative double staining for Bim (red, f) or Pdx1 (red, g) and insulin (green) from MLD-STZ–treated Mst1^−/− mice and controls killed at day 22. White arrows indicate areas of cytosolic Pdx1 localization and its total absence in WT-STZ–treated mice. Scale bars, 100 Mm.

(h–j) bMst1^−/− mice with specific deletion in the beta cells using the Cre-loxP system (n = 5) and RIP-Cre (n = 3) and Mst1^fl/fl controls (n = 3) injected with 40 mg per kg body weight STZ for five consecutive days. (h) Random fed blood glucose measurements after last STZ injection (day 0) over 32 d and i.p. GTT at day 30. (i) Insulin secretion during an i.p. GTT measured before (0 min) and 30 min after glucose injection. Data are expressed as ratio of secreted insulin at 30 min to that secreted at 0 min (stimulatory index). The ratio of secreted insulin and glucose calculated at fed state (right).

(j) Beta cell mass, TUNEL or Ki-67 analysis, expressed as percentage of TUNEL- or Ki-67–positive beta cells from mice at day 32. Data show means o s.e.m. *P < 0.05 MLD-STZ–treated WT mice compared to saline-injected WT mice, **P < 0.05 MLD-STZ–treated MST1^−/− mice compared to MLD-STZ–treated WT mice. ^#P < 0.05 MLD-STZ–treated bMst^−/− mice compared to MLD-STZ–treated Mst1^fl/fl or RIP-Cre mice.

Im Dokument beta-MSCs: Successful fusion of bone marrow mesenchymal stromal cells with beta-cells results in a beta-cell like phenotype (Seite 116-188)